1. Field of the Invention
The present invention relates to the isolation and purification of a proteinase inhibitor, and more specifically, to a method for controlling the yield and purity of Proteinase Inhibitor-II (PI2) extracted from whole potatoes by adjusting heat treatment and filtration conditions.
2. Background of the Prior Art
Proteins that inhibit proteolytic enzymes are often found in high concentrations in many seeds and other plant storage organs. Inhibitor proteins are also found in virtually all animal tissues and fluids. These proteins have been the object of considerable research for many years because of their ability to complex with and inhibit proteolytic enzymes from animals and microorganisms. The inhibitors have become valuable tools for the study of proteolysis in medicine and biology. Protease inhibitors are of particular interest due to their therapeutic potentials in controlling proteinases involved in a number of disorders such as pancreatitis, shock, and emphysema, and as agents for the regulation of mammalian fertilization. Potato tubers are a rich source of a complex group of proteins and polypeptides that potently inhibit several proteolytic enzymes usually found in animals and microorganisms. In particular, potato inhibitors are known to inhibit human digestive proteinases, and thus have application in the control of obesity and diabetes.
Two broad classes of protease inhibitor superfamilies have been identified from soybean and other legumes with each class having several isoinhibitors. Kunitz-type inhibitor is the major member of the first class whose members have 170-200 amino acids, molecular weights between 20,000 and 25,000, and act principally against trypsin. Kunitz-type proteinase inhibitors are mostly single chain polypeptides with 4 cysteines linked in two disulfide bridges, and with one reactive site located in a loop defined by disulfide bridge. Kunitz inhibitor is capable of inhibiting trypsin derived from a number of animal species as well as bovine chemotrypsin, human plasmin, and plasma kallikrein. The cationic form of human trypsin, which accounts for a majority of trypsin activity, is only weakly inhibited by the Kunitz inhibitor, whereas the anionic form is fully inhibited.
The second class of inhibitors contains 60-85 amino acids, has a range in molecular weight of 6000-10,000, has high proportion of disulfide bonds, is relatively heat-stable, and inhibits both trypsin and chemotrypsin at independent binding sites. Bowman-Birk inhibitor is an example of this class. The Bowman-Birk inhibitor is a 71 amino acid chain protein with 7 disulfide bonds characterized by its low molecular weight of about 8000 (in non-associated monomers), high concentration (about 20%) of cystine, high solubility, resistance to heat denaturation and having the capacity to inhibit trypsin and chymotrypsin at independent inhibitory sites.
Proteinase inhibitors extracted from potatoes have been distinguished into two groups based on their heat stability. The group of inhibitors that is stable at 80° C. for 10 minutes have been identified as inhibitor I (mol. wt. 39,000) (Melville, J. C. and Ryan, C. A. Chymotrypsin inhibitor I from potatoes. J. Biological Chem., 247: 3445-3453, 1972), carboxypeptidase inhibitor (CPI) (mol. wt. 4,100) (Ryan, C. L., Purification and properties of a carboxypeptidase inhibitor from potatoes. J. Biol. Chem. 249: 5495-5499, 1974), inhibitors IIa and IIb (mol. wt. 20,700) (Bryant, J., Green, T. R., Gurusaddaiah, T., Ryan, C. L. Proteinase inhibitor II from potatoes: Isolation and characterization of its protomer components. Biochemistry 15: 3418-3424, 1976), and inhibitor A5 (mol. wt. 26,000).
In 1972, Melville and Ryan (Melville et al.) reported a large-scale preparation for isolating Chymotrypsin Inhibitor I from potato tubers. According to the method of Melville and Ryan, potatoes were sliced with peels intact and soaked in a sodium dithionite solution, homogenized, and expressed through nylon cloth. The resulting juice was adjusted to a pH of 3, centrifuged at 1000×g for 15 minutes at 5° F., and the supernatant collected and fractionated with ammonium sulfate.
Purification was achieved through water washing and heat treatment whereby clear filtrates of heated fractions were pooled and lyophilized. Suspending the lyophilized powder in water, dialyzing it against water for 48 hours, and lyophilizing the resulting clear filtrate obtained a crude extract. Resuspended extract was then centrifuged and applied to a column of Sephadex G-75. Collected fractions containing the Inhibitor I were pooled, evaporated, and desalted on a column of Sephadex G-25. The resulting gel-filtered inhibitor product was determined to be approximately 90% Inhibitor I protein purified by dissociation on a Sephadex G-75 column and desalted on a column of Sephadex G-25.
The Ryan lab followed-up by reporting the isolation and characterization of Proteinase Inhibitor II in much the same manner as described for Inhibitor I (Bryant, J., Green, T. R., Gurusaddaiah, T., Ryan, C. L. Proteinase inhibitor II from potatoes: Isolation and characterization of its protomer components. Biochemistry 15: 3418-3424, 1976). Bryant et al. differentiated potato-derived proteinase inhibitors into two groups based on their respective stabilities to a temperature of 80° C. for 10 minutes. Proteinase Inhibitor I (PI1) is characterized as a tetrameric protein composed of four hybridized isoinhibitor protomer species having a molecular weight of 39,000, whereas PI2 is characterized as a dimeric inhibitor comprising four isoinhibitor promoter species having a molecular weight of 21,000.
The isolation of proteinase inhibitor proteins from potatoes is described in WO 99/01474. Proteins from potato tubers are extracted in soluble form in an aqueous/alcohol extraction medium, such as dilute formic acid and 20% ethanol. The alcohol extract is heated to a first temperature to denature most of the unwanted proteins and cooled to a second temperature to form a precipitate phase constituting the debris and a soluble phase that contains the heat stable proteinase inhibitor proteins. The heat stable proteinase inhibitor proteins are precipitated from the soluble phase by dialysis against a suitable dialysis medium, such as dilute formic acid.
U.S. Pat. No. 5,187,154 describes a method for the diagnosis and the treatment of individuals with diabetes or at risk to develop diabetes mellitus. In particular, gastric emptying determinations are used to assess risk. Risk or early symptoms associated with subsequent development of diabetes mellitus may be controlled or alleviated by delaying gastric emptying, which was achieved by the administration of cholecystokinin.
U.S. Pat. No. 4,906,457 describes compositions and methods for reducing the risk of skin cancer. The described compositions included at least one effective protease inhibitor. Preferred protease inhibitors included serine protease inhibitors and metallo-protease inhibitors. The protease inhibitors were preferably included in concentrations ranging from approximately 10 picograms to 10 milligrams per milliliter of the skin-applicable topical mixtures. The topical mixtures preferably included a suitable topical vehicle such as a cream, lotion, or ointment. One class of anti-carcinogenic skin treatment compositions of this invention preferably included the desired protease inhibitors in combination with a suitable sunscreen agent or agents, such as para-amino benzoic acid, to provide particularly advantageous compositions for reducing the risk of sunlight-induced skin cancer.
When applied to mouse epidermal JB6 cells, proteinase inhibitors I and II from potatoes blocked the UV induced transcription factor activator protein-1 (AP-1), which has been shown to be responsible for the tumor promoter action of UV light in mammalian cells. The inhibition appears to be specific for UV induced signal transduction for AP-1 activation. Furthermore, the inhibition of UV induced AP-1 activity occurs through a pathway that is independent of extracellular signal-regulated kinases and c-jun N-terminal kinases as well as P38 kinases (Huang, C., Ma, W. Y., Ryan, C. A., Dong, Z. Proteinase inhibitors I and II from potatoes specifically block UV-induced activator protein-I activation through a pathway that is independent of extracellular signal regulated kinases, c-jun N-terminal kinases, and P38 kinase. Proc. Natl. Acad. Sci., US, 94: 11957-11962, 1997).
U.S. Pat. No. 4,491,578 describes a method of eliciting satiety in mammals through the administration of an effective amount of a trypsin inhibitor. The method was based on the postulate that the enzyme trypsin, normally secreted by the pancreas, constitutes a negative feedback signal for cholecystokinin secretion that in turn comprises a putative satiety signal. Thus, the effect of the trypsin inhibitor is to increase the concentration of cholecystokinin secretion advancing the sensation of satiety resulting in a consequent decrease in food intake and, over time, body weight.
The effect of PI2 extracted from potatoes, which increases CCK release, on food intake was examined in 11 lean subjects. Five minutes before presenting them with a lunchtime test meal, volunteers received 1.5 g PI2 in a high protein soup vehicle (70 kcal), the soup vehicle alone, or a no-soup control, according to a double blind, within subject design. The consumption of the soup alone led to a non-significant 3% reduction in energy intake. The addition of 1.5 g PI2 to the soup significantly reduced energy intake by additional 17.5%. Pre-meal ratings of motivation to eat and food preferences did not predict the reduction in energy intake by the proteinase inhibitor. Based on the results, the authors concluded that endogenous CCK may have an important role in the control of food intake and that proteinase inhibition may have a potential for reducing food intake (Hill et al., 1990).
The efficiency of oral ingestion of trypsin/chemotrypsin inhibitor in delaying the rate of gastric emptying in recently diagnosed type II diabetic patients and improving their post-prandial metabolic parameters have been examined (Schwartz, J. G., Guan, D., Green, G. M., Phillips, W. T.). Treatment with an oral proteinase inhibitor slows gastric emptying and actually reduces glucose and insulin levels after a liquid meal in type II diabetic patients. Diabetes Care, 17: 255-262, 1994). Serum insulin, plasma glucose, plasma gastric inhibitory polypeptide levels, and the rate of gastric emptying were all significantly decreased over the 2 hour testing period in subjects who received proteinase inhibitor in their oral glucose/protein meal. U.S. Pat. No. 5,187,154 suggests that the administration of CCK can be made through an intramuscular injection or an intranasal spray. Alternatively, an oral administration of an agent that enhances endogenous release of CCK could represent an important approach to the treatment of Type 2 diabetes. One of the agents that may have a therapeutic application in patients with recently diagnosed Type 2 diabetes can be the potato proteinase inhibitor II.
Recently, PI2 has been implicated in playing a role in extending satiety in subjects fed a nutritional drink composition containing PI2. U.S. patent application Ser. No. 09/624,922 describes that subjects reported a significant reduction in hunger for up to 3½ hours post meal when fed a meal comprising a nutritional drink composition containing PI2. Likewise, fullness ratings were enhanced, and each study subject lost an average of 2 kg over a 30-day period without experiencing the adverse side effects typically associated with appetite suppressing compounds. Mechanistically, it is thought that as a trypsin and chymotrypsin inhibitor, when consumed by a subject, PI2 stimulates the release of endogenous cholecystokinin, a known putative feedback agent effective in reducing the desire to intake food.
Proteinase inhibitor II is seen to have potential applicability in a number of areas affecting human health. In addition, PI2 may be administered in a variety of ways, including orally, intramuscularly, intranasally, and topically, and will be provided in a variety of carriers and diluents. In certain applications, for example in the control of satiety, it has been found that a relatively pure form of PI2 is needed and that the presence of impurities, such as Kunitz inhibitors, adversely affects the efficacy of the PI2. In other applications, purity will likely not be as critical so that the extraction process could be simplified, thereby reducing the cost of the PI2 product. Accordingly, a need exists for a large-scale isolation and purification process to extract PI2 in a cost-effective and efficient manner meeting commercial qualitative and quantitative standards.
A technique capable of large-scale isolation and purification is ultrafiltration, a type of membrane filtration and separation technique that utilizes membranes having pore sizes between 0.001 and 0.1 μm. Methodologies utilizing ultrafiltration are particularly useful for concentrating dissolved molecules such as proteins, peptides, nucleic acids, carbohydrates, and other biomolecules, as well as desalting, exchanging buffer, and gross fractionation. Diafiltration is a selective fractionation process of washing smaller molecules through a membrane, while leaving the larger molecule of interest in the retained solution, also known as retentate. In selecting a membrane suitable for filtering a target molecule, the molecular weight cutoff (MWCO) of a membrane is utilized to define the ability of the membrane to exclude molecules on a size basis. 90% of an ideally globular molecule. MWCO is the size designation (in kilodaltons “KD”) for ultrafiltration membranes. The term Nominal Molecular Weight Cutoff (NMWCO) is defined as a membrane's ability to retain 90% of an ideally globular molecule having the designated molecular weight.
As discussed above, ultrafiltration is a technique used for the separation under elevated pressure of dissolved molecules in solution on the basis of size. Molecules larger than the pore size of the membrane will not pass through the membrane surface and will remain in the retentate, and may further be retained on the surface of the membrane. Accumulations of retained molecules on the membrane typically form a gel layer, significantly reducing the separation performance characteristics of the membrane. Separation capacity of a given ultrafiltration system is a function of the ability of the selected membrane to allow the smaller particles to pass through the membrane in the permeate, while minimizing gel formation in the retentate. However, a direct correlation between a molecular weight and size does not always exist.
Molecular conformations (including both intermolecular and intramolecular interactions) can significantly alter the apparent size of a molecule. Having numerous modes of interaction available, proteins are particularly susceptible to conformational changes while dissolved in solution. Solution characteristics such as pH, solute concentration, temperature, and ionic strength significantly affect the apparent size of particles and molecules in solution, and therefore affect conformational characteristics as well.